Laboratory 9
Enumeration
Introduction
One of the central questions a researcher asks when studying any bacterial system is
“how many bacteria are present in this sample?” This applies to environmental samples where
we want to learn about a native population as well as to lab cultures where we are measuring
physiological functions. There are many ways to count bacteria and each of them has advantages
and disadvantages.
Measuring the optical density (O.D.) of a sample is an indirect method of determining the
number of cells present. The amount of light of a specific wavelength that is absorbed by a
culture is related to the number of cells. This is a very fast and easy way to count cells when
possible. Unfortunately, in order to use this method, you first need to calculate a response factor
(the ratio of the number of cells to the change in absorbance) for the specific cells and media that
you are using. This requires use of an independent method for counting the cells.
Another common method for counting cells is to serially dilute the original sample and
spread-plate the resulting dilutions. This is known as the plate-count method. The colonies that
arise after incubation are counted to determine the original cell density. This method requires
more time and supplies than measuring the optical density but it does result in an absolute
number of cells rather than a relative value. The one important thing to remember when doing
plate-counts is that it only counts the viable cells (those cells that are able to grow under the
specific incubation conditions used).
If you want to count all of the cells present in a sample then a direct-counting method
should be chosen. This generally involves using a microscope to visualize the cells. Special
microscope slides that have surfaces calibrated into precise volumes can be used to determine
exactly how many cells there are in a milliliter of sample. Sometimes stains are used to make the
cells more visible. This method counts all cells whether they are alive or not. The decision as to
whether viable or total cell counts are more relevant will depend on the experimental question
being asked.
In this lab you will be counting the number of bacteria in an overnight culture of E. coli
using three different methods: optical density, direct counts and plate counts. You will then be
able to calculate a response factor for optical density and compare the direct count and viable
count methods.
Materials
Equipment
- spectrophotometer
- microscopes
- Petroff-Hauser chamber
- bunsen burners
- spreaders
Cultures
- Escherichia coli (broth)
Supplies
- Nutrient Agar (NA) plates
- Liquid NB media
- Pasteur pipettes and bulbs
- Methylene Blue stain
- 95% ethanol
Procedures
Plate-Counts (Counting by Dilution Series)
Period 1
1. Make a dilution series of the E. coli culture in liquid NB media.
a. Aseptically transfer 1 ml of the culture into a tube containing 9 ml of NB medium.
b. Transfer 1 ml of the resulting tube into another tube with 9 ml of medium.
c. Repeat this process until you have produced 8 tubes. (10-1 – 10-8).
2. Spread 0.1 ml of each dilution tube from 10-2 – 10-8 onto a NA plate.
3. Incubate at 35C for one day.
Period 2
4. Count the number of colonies on plates that contain between 20 and 200 wellseparated colonies.
5. Calculate the number of cells in the starting culture by multiplying the number of
colonies times the inverse of the dilution.
Optical Density (Counting by Spectrophotometer)
1. Turn on the spectrophotometer and set it to 525 nm.
2. Make a dilution series of the original E. coli culture in liquid NB media.
a. Transfer 5 ml of the culture into a tube containing 5 ml of NB medium.
b. Transfer 5 ml of the resulting tube into another tube with 5 ml of medium.
c. Repeat this process until you have produced 4 new tubes. (1:2 – 1:16).
3. Set the transmittance to 0 with no sample in the chamber.
4. Set the absorbance to 0 using un-inoculated NB medium as a blank.
5. Measure the absorbance of each of the dilution tubes used in the dilution series.
6. Plot the absorbance against the dilution factor.
Direct Counting (Petroff-Hauser Chamber)
1. Use a pipette to transfer 0.9 ml of the starting culture to a clean test-tube.
2. Add 0.1 ml of methylene blue stain to the culture.
3. Use a Pasteur pipette to transfer a drop of the stained culture into the Petroff-Hauser
slide.
4. Carefully place the cover slip onto the calibrated surface of the counting chamber.
5. Place the chamber on the microscope stage and focus under the 20X objective to
locate the large counting grids.
6. Switch to high power and count the number of bacteria in at least 20 of the small
squares.
(If there are too many bacteria to count, make a 1:10 dilution of the culture and start
over)
7. Average the number of cells and calculate the number per ml. (cubic centimeter).
Dilution Series
Lab #9
20 points
Name:
Date:
Plate counts
Count the number of colonies on plates that contain between 20 and 200 well-separated
colonies.
colonies
dilution
Calculate the number of cells in the starting culture by multiplying the number of colonies
times the inverse of the dilution.
Concentration of cells in starting culture =
Optical density
dilution tube
absorbance
culture
1:2
1:4
1:8
1:16
Plot the absorbance against the dilution factor. (Attach graph)
cells/ml
Direct counts
Average the number of cells/square and calculate the number per ml. (cubic centimeter).
Each small square is 1/20th X 1/20th of one millimeter square and 1/50th of one millimeter
deep.
Average number of cells per square Cells per milliliter Cells per milliliter -
(corrected for dilution by dye)
Response factor
Use the absorbance graph, the plate-count data and the direct-count data to calculate two
separate response factors (# of cells / absorbance unit) for absorbance at 620nm for E. coli.
Plate count response factor
cells/absorbance unit
Direct count response factor
cells/absorbance unit
Question:
1). Which response factor is more accurate for correlating absorbance to cell numbers? Why?
2). Give an example of an experiment in which you would want to use a direct count and an
example of an experiment in which you would want to use a live count for quantifying bacteria.